The proposed study will focus on matrix vesicles (MVs) which play an initiating role in mineralization of teeth and bones. MVs are submicroscopic extracellular, membrane-invested particles that serve as the initial site of calcification in dentin, growth plate cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We and others have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), and ATPase are involved in MV mineralization. Furthermore, these phosphatase are integrated into the MV membrane which mineral first appears, suggesting a critical role for components of the MV membrane in initiating calcification. Our specific aims are directed toward an increased understanding of the molecular and structural organization of the MV membrane and sap with emphasis on identify and localizing major constitutive proteins, testing the function of MVs, and studying the regulation of MV biogenesis by cultured bone or cartilage progenitor cells. Specific aims: 1) Identification of major MV proteins that may play a role in calcification, including non-collagenous proteins of bone (bone sialoprotein, osteopontin, osteonectin, osteocalcin), calpain, and P-glycoprotein. 2) Experimental calcification of isolated MVs to further elucidate the role of vesicle phosphatases, especially ALP and ATPase, plus other integral MV proteins in the calcification mechanism. 3) a study of the mechanism of MV biogenesis by plasma membrane budding, examining possible regulators of MV biogenesis (e.g. the bone morphogenetic proteins) and the relation of MV biogenesis to differentiation and programmed cell death (apoptosis). This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.